The present application is directed to a method of quantitative analysis of a macromolecule which is bindable by a macromolecular specific binding partner, by capillary electrophoresis.
For many purposes, it is necessary to know the amount of a target macromolecule in a sample, where the target macromolecule is similar in its chemical and biophysical properties, to another macromolecule in the sample. The similar macromolecules involved can differ in their primary structure, such as isotopes of antibodies, or can be different as the result of reactions in which only one of the two macromolecules is changed.
A clinically important example of the latter is glycosylated hemoglobin A. Human adult hemoglobin (Hb) typically consists of Hb A, Hb A.sub.2, and Hb F. These forms of hemoglobin differ by virtue of their primary structure; that is, by their amino acid sequence. Normally, Hb A constitutes about 97% of the total hemoglobin present, Hb A.sub.2 constitutes about 2.5% of the total, and Hb F only about 0.5%. Hb F is also referred to as fetal hemoglobin and is the normal form of hemoglobin found before birth. Shortly after birth, the normal adult form Hb A begins to predominate. In some disorders, Hb F persists. In other disorders, of which the most common is sickle cell syndrome, an abnormal hemoglobin occurs where a different amino acid sequence replaces Hb A. In sickle cell syndrome, this abnormal hemoglobin is known as Hb S.
Chromatographic analysis of Hb A has shown that it contains a number of minor hemoglobin species. Bisse and Wieland, "High Performance Liquid Chromotographic Separation of Human Hemoglobins," J. of Chromotography, 434 (1988) 95-110. These minor species have been designated Hb A.sub.1a, Hb A.sub.1b, and Hb A.sub.1c. These species are referred to as glycosylated hemoglobins or glycohemoglobins, and are formed by condensation of the amino group of the hemoglobin with a keto moiety of a sugar. For Hb A.sub.1c, the sugar is glucose, and the glycosylated hemoglobin is formed by the condensation of the N-terminal valine amino acid of each .beta.-chain of the hemoglobin with glucose to form an unstable Schiff base or aldimine (also known as pre-A.sub.1c), which then undergoes an Amadori rearrangement to form a stable ketoamine.
The formation of glycosylated hemoglobins is nonenzymatic. It occurs over the lifespan of the red cell, which is about 120 days under normal conditions. It is also proportional to the concentration of glucose in the blood. The amount of Hb A.sub.1c in blood is therefore related to time-averaged glucose concentration over the period of two or three months prior to the measurement. This value provides a way of assessing the control of diabetes, where the results are not affected by short-term fluctuations in plasma glucose levels. Therefore, measurement of glycohemoglobins can supplement other more traditional methods of assessing control of diabetes. For example, measurement of glycohemoglobins can be used when urine glucose records are inadequate or cannot be maintained, when blood glucose levels vary markedly throughout the day or from day to day, and for a new patient with known or suspected diabetes in whom there is no previous record of blood glucose concentration. A particular application for monitoring glycohemoglobins is during pregnancy, when close control of diabetes is especially important.
Currently available methods for the determination of glycohemoglobin include ion exchange chromatography, high-performance liquid chromatography, affinity chromatography, colorimetry, radioimmunoassay, electrophoresis, and isoelectric focusing. A comparison of these methods has been reported. Goldstein, et al., "Recent Advances in Glycosylated Hemoglobin Measurements," C.R.C. Critical Reviews in Clinical Laboratory Sciences, 21 (3), pp. 187-228.
Ion exchange chromatography can be carried out using resins containing weakly acidic cation exchanges or negatively charged carboxymethylcellulose resin. This procedure is time consuming and requires rigid control of temperatures of the reagents and the columns as well as the pH and the ionic strength. In practice, this means that these methods are usable only by highly skilled personnel and are not well suited to routine clinical determinations.
High performance liquid chromatography, although reliable as a reference method, also requires close control of pH and ionic strength as well as other variables.
Affinity chromatography can be used to separate nonglycosylated hemoglobin from glycosylated hemoglobin. A suitable affinity column is prepared from a gel containing immobilized m-aminophenylboronic acid on cross-linked, beaded agarose. The boronic acid reacts with the cis-diol groups of glucose bound to hemoglobin to form a reversible 5-membered ring complex, thus selectively binding the glycosylated hemoglobin to the affinity column. The nonglycosylated hemoglobin passes through the column. The glycosylated hemoglobin is then dissociated from the complex by sorbitol. Although this method is more precise, being less susceptible to variations in temperature or ionic conditions than is ion-exchange chromatography, the affinity columns must be protected from sunlight and can only be reused a limited number of times before they must be discarded.
A calorimetric method has been devised based on the observation that Hb A.sub.1c, when subject to mild acid hydrolysis, releases 5-hydroxymethylfurfural (5-HMF). This test has proven difficult to standardize because the yield of 5-HMF from Hb A.sub.1c is only about 30%. In order to provide reliable results, reaction conditions must be carefully controlled. Therefore, this method is unsuitable for routine clinical analysis, particularly when rapid results are needed.
Another spectrophotometric method involves the reaction of inositol hexaphosphate (phytic acid) with hemoglobin. When phytic acid is added to a solution of hemoglobin, a shift in the absorption spectrum occurs in the visible region, as phytic acid binds to the N-terminal amino groups of the .beta.-chains. Absorbance increases at 633 nm and decreases at 560 nm. This change only occurs for Hb A that is unglycosylated. The spectrum of glycosylated hemoglobin is not changed because the blocking effect of the glucose moiety prevents binding of phytic acid to the N-terminal amino groups of the .beta.-chains. The change in absorbance induced by phytic acid is thus inversely proportional to the fraction of glycosylated hemoglobin. This observation can be used as the basis of a spectrophotometric assay. However, the compound 2,3-diphosphoglycerate (DPG), normally present in red cells, binds to the same region of the hemoglobin molecule as phytic acid and reduces the available sites for phytic acid. This leads to higher apparent results of glycosylated hemoglobins. Because endogenous DPG concentrations are variable and are not normally known for a given sample, the method therefore has limited applicability.
Antibody against Hb A.sub.1c can be prepared and used as the basis for a radioimmunoassay. Guthrie, et al., "A Multisite Physician's Office Laboratory Evaluation of an Immunological Method for the Measurement of HbA.sub.ic," Diabetes Care, Vol. 15, No. 11, 11/92, 1494-8. However, such a radioimmunoassay, like radioimmunoassays in general, brings with it the problems of the disposal of reagents and the short shelf life of reagents due to degradation caused by radioactive labeling, with consequent loss of specific reactivity. Thus, radioimmunoassay, though capable of accuracy, cannot generally be used for routine determinations of Hb A.sub.1c.
Agarose gel electrophoresis is a time-consuming and tedious method.
Isoelectric focusing has also been used as a method of quantitating Hb A.sub.1c. Although the method works well, it is cumbersome and requires accurate performance to ensure that the pH range established by the ampholytes is reproducible. Molten, et al., "Application of Dynamic Capillary Isoelectric Focusing to the Analysis of Human Hemoglobin Variants," Electrophoresis (1994) 15, 22-30.
In view of the shortcomings of these various methods, there is a need for an improved method that can differentiate two similar macromolecules from each other, and particularly distinguish Hb A.sub.1c from other forms of hemoglobin. Preferably, the method is rapid and reproducible and can be used in the clinical laboratory to aid in rapid diagnosis and to evaluate courses of treatment. Preferably, such a method reduces or eliminates the risk of operator error and does not use toxic reagents such as acrylamide solutions or radioactive reagents.